The goals of this research are to delineate the cis-acting signals, identify the trans-acting factors and elucidate the mechanisms that regulate human cytomegalovirus (CMV) gene expression at the translational level. Investigations of the molecular basis of CMV translational control will reveal both viral-specific processes, potentially useful as targets for antiviral interventions, as well as new insights into fundamentals of eukayrotic translation. This project focuses on the CMV gpUL4 protein, synthesis of which is regulated by a short upstream open reading frame (uORF2) in the gpUL4 mRNA. Mutant viruses will be used to evaluate the effects of uORF2 alterations on regulation of gpUL4 expression and on viral replication. In conjunction with investigations of the consequences of ectopic gpUL4, these studies will illuminate the significance of gpUL4 translational regulation in the viral life cycle. A second series of experiments will elucidate the detailed molecular mechanism by which uORF2 regulates gene expression. Genetic studies using S. cerevisaie, complemented by molecular biological and biochemical assays in mammalian cells and cell extracts will delineate the precise step during translation termination that is controlled by uORF2 and will identify trans-acting factors that participate in this extraordinary regulatory mechanism.